Plant oils with altered oleic acid content

ABSTRACT

By this invention, methods to produce oleic fatty acids in plant seed oils are provided. The methods of the present invention generally involve the suppression of a host plant cells endogenous β-ketoacyl-ACP synthase I protein. Also described in the instant invention are the plants, cells and oils obtained therefrom.

This application is a continuation of application Ser. No. 09/304,603filed May 3, 1999 now U.S. Pat. No. 6,483,008, which is acontinuation-in-part of application Ser. No. 07/987,256 filed Dec. 7,1992 now U.S. Pat. No. 6,348,642, which is a continuation-in-part ofapplication Ser. No. 07/568,493 filed Aug. 15, 1990, now abandoned, eachof which is herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of thesequence listing on diskette as filed on Nov. 11, 2000 in the parentapplication, application Ser. No. 09/304,603 filed May 4, 1999, areherein incorporated by reference.

INTRODUCTION

1. Field of Invention

The present invention is directed to methods for the increasedproduction of particular fatty acids in plants. In particular, thepresent invention is directed to methods for increasing oleic acid inplants.

2. Background

Plant oils are used in a variety of industrial and edible uses. Novelvegetable oils compositions and/or improved means to obtain oilscompositions, from biosynthetic or natural plant sources, are needed.Depending upon the intended oil use, various different fatty acidcompositions are desired.

For example, in some instances having an oilseed with a higher ratio ofoil to seed meal would be useful to obtain a desired oil at lower cost.This would be typical of a high value oil product. In some instances,having an oilseed with a lower ratio of oil to seed meal would be usefulto lower caloric content. In other uses, edible plant oils with a higherpercentage of unsaturated fatty acids are desired for cardio-vascularhealth reasons. And alternatively, temperate substitutes for highsaturate tropical oils such as palm and coconut, would also find uses ina variety of industrial and food applications.

One means postulated to obtain such oils and/or modified fatty acidcompositions is through the genetic engineering of plants. However, inorder to genetically engineer plants one must have in place the means totransfer genetic material to the plant in a stable and heritable manner.Additionally, one must have nucleic acid sequences capable of producingthe desired phenotypic result, regulatory regions capable of directingthe correct application of such sequences, and the like. Moreover, itshould be appreciated that in order to produce a desired phenotyperequires that the Fatty Acid Synthetase (FAS) pathway of the plant ismodified to the extent that the ratios of reactants are modulated orchanged.

Higher plants appear to synthesize fatty acids via a common metabolicpathway. In developing seeds, where fatty acids are attached to glycerolbackbones, forming triglycerides, are stored as a source of energy forfurther germination, the FAS pathway is located in the proplastids. Thefirst committed step is the formation of acetyl-ACP (acyl carrierprotein) from acety-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACPtransacylase (ATA). Elongation of acetyl-ACP to 16- and 18-carbon fattyacids involves the cyclical action of the following sequence ofreactions: condensation with a two-carbon unit from malonyl-ACP to forma β-ketoacyl-ACP (β-ketoayl-ACP synthase), reduction of theketo-function to an alcohol (β-ketoacyl-ACP reductase), dehydration toform an enoyl-ACP (β-hydroxyacyl-ACP dehydrase), and finally reductionof the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACPreductase). β-ketoacyl-ACP synthase I, catalyzes elongation up topalmitoyl-ACP (C16:0), whereas β-ketoacyl-ACP synthase II catalyzes thefinal elongation to stearoyl-ACP (C 18:0). Common plant unsaturatedfatty acids, such as oleic, linoleic and a-linolenic acids found instorage triglycerides, originate from the desaturation of stearoyl-ACPto form oleoyl-ACP (C18:1) in a reaction catalyzed by a soluble plastidΔ-9 desaturase (also often referred to as “stearoyl-ACP desaturase”).Molecular oxygen is required for desaturation in which reducedferredoxin serves as an electron co-donor. Additional desaturation iseffected sequentially by the actions of membrane bound Δ-12 desaturaseand Δ-15 desaturase. These “desaturases” thus create mono- orpolyunsaturated fatty acids respectively.

A third β-ketoacyl-ACP synthase has been reported in S. oleracea leaveshaving activity specific toward very short acyl-ACPs. This acetoacyl-ACPsynthase or “β-ketoacyl-ACP” synthase III has a preference to acetyl-CoAover acetyl-ACP, Jaworski, J. G., et al., Plant Phys. (1989) 90:41-44.It has been postulated that this enzyme may be an alternate pathway tobegin FAS, instead of ATA.

Obtaining nucleic acid sequences capable of producing a phenotypicresult in FAS, desaturation and/or incorporation of fatty acids into aglycerol backbone to produce an oil is subject to various obstaclesincluding but not limited to the identification of metabolic factors ofinterest, choice and characterization of an enzyme source with usefulkinetic properties, purification of the protein of interest to a levelwhich will allow for its amino acid sequencing, utilizing amino acidsequence data to obtain a nucleic acid sequence capable of use as aprobe to retrieve the desired DNA sequence, and the preparation ofconstructs, transformation and analysis of the resulting plants.

Thus, the identification of enzyme targets and useful plant sources fornucleic acid sequences of such enzyme targets capable of modifying fattyacid compositions are needed. Ideally an enzyme target will be amenableto one or more applications alone or in combination with other nucleicacid sequences, relating to increased/decreased oil production, theratio of saturated to unsaturated fatty acids in the fatty acid pool,and/or to novel oils compositions as a result of the modifications tothe fatty acid pool. Once enzyme target(s) are identified and qualified,quantities of purified protein and purification protocols are needed forsequencing. Ultimately, useful nucleic acid constructs having thenecessary elements to provide a phenotypic modification and plantscontaining such constructs are needed.

SUMMARY OF THE INVENTION

The present invention is directed to methods for producing plant oilshaving elevated levels of oleic acid (C18:1) as a percentage of thetotal fatty acids.

The method generally comprises growing a plant containing a constructhaving as operably linked components in the 5′ to 3′ direction oftranscription, a promoter region functional in a host plant cell, atleast a portion of a nucleic acid sequence encoding a β-ketoacyl ACPsynthase in an antisense orientation and a transcription terminationsequence.

The methods described herein are utilized to produce plants withincreased levels of oleic acid. Increases of at least 5 percent to 60percent, preferably, 10 percent to 50 percent, more preferably 10percent to 40 percent over the wild type seed oil are encompassed by themethods provided herein.

In one embodiment of the present invention, a Brassica seed oil havingincreased oleic acid is obtained using the methods of the presentinvention. The oleate content of the Brassica seed oil preferablycomprises greater than 65%, more preferably greater than about 75% ofthe fatty acid moieties in the oil. The oil of the present invention maybe used as a blending source to make a blended oil product, or it mayalso be used in the preparation of food.

In another embodiment of the present invention, a Brassica oil having adecreased polyunsaturated fatty acid composition is obtained using themethods described herein. Brassica oils with polyunsaturated fatty acidcompositions of less than about 12 weight percent are exemplifiedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D: The fatty acid composition from about 50 individual seedsfrom each of two lines of plants containing the construct pCGN3259.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, constructs and methods areprovided for the production of plants with an increased level of Oleicacid (C18:1), as a percentage of the total fatty acids. The methods forproducing such plants generally comprise transforming a host plant cellwith expression constructs having a promoter sequence functional in aplant operably associated to at least a portion of a nucleic acidsequence encoding a β-ketoacyl-ACP synthase (referred to herein as KAS)in an anti-sense orientation, and a transcription termination sequence.The expression constructs provide a novel method to increase in thelevels of oleic acid in the seed oil of the transformed plants.

β-ketoacyl-ACP synthases are well known in the art for their involvementin the biosynthesis of fatty acids. The first step in the biosynthesisof fatty acids is the formation of acetyl-ACP (acyl carrier protein)from acetyl-CoA and ACP catalyzed by a short chain preferring condensingenzyme, β-ketoacyl-ACP synthase (KAS) III. Elongation of acetyl-ACP to16- and 18-carbon fatty acids involves the cyclical action of thefollowing sequence of reactions: condensation with a two-carbon unitfrom malonyl-ACP to form a longer β-ketoacyl-ACP (β-ketoacyl-ACPsynthase), reduction of the keto-function to an alcohol (β-ketoacyl-ACPreductase), dehydration to form an enoyl-ACP (β-hydroxyacyl-ACPdehydrase), and finally reduction of the enoyl-ACP to form the elongatedsaturated acyl-ACP (enoyl-ACP reductase). β-ketoacyl-ACP synthase I (KASI), is primarily responsible for elongation up to palmitoyl-ACP (C16:0),whereas β-ketoacyl-ACP synthase II (KAS II) is predominantly responsiblefor the final elongation to stearoyl-ACP (C18:0).

Genes encoding peptide components of β-ketoacyl-ACP synthases I and IIhave been cloned from a number of higher plant species, including castor(Ricinus communis) and Brassica species (U.S. Pat. No. 5,510,255). KAS Iactivity was associated with a single synthase protein factor having anapproximate molecular weight of 50 kD (synthase factor B) and KAS IIactivity was associated with a combination of two synthase proteinfactors, the 50 kD synthase factor B and a 46 kd protein designatedsynthase factor A. Cloning and sequence of a plant gene encoding a KASIII protein has been reported by Tai and Jaworski (Plant Physiol. (1993)103:1361-1367).

Surprisingly, it is found herein that the antisense expression of atleast a portion of a KAS I sequence in the seed cells of a host plantcell increases the oleic acid content of the seed oil.

Preferably, the KAS sequences used in the present invention are derivedfrom the endogenous KAS sequence of the target host plant, also referredto herein as the native KAS sequence. The skilled artisan will recognizethat also of use in the present invention are non-native KAS sequencesobtained from sources other than the target host plant. By target hostplant is meant the plant into which the expression constructs containingthe KAS sequences are transformed.

As described in more detail in the examples that follow, a β-ketoacylACP synthase type I (referred to herein as KASI) coding sequence fromBrassica (U.S. Pat. Nos. 5,475,099, and 5,510,255, the entireties ofwhich are incorporated herein by reference) is used in expressionconstructs in an antisense orientation to generate transgenic Brassicaplants with decreased production of the KASI in host cells.

Surprisingly, it is demonstrated herein that transformation of a plantwith a construct providing antisense expression of the KASI gene leadsto a significant increase in the levels of oleic acid (C18:1) obtainedas a percentage of the total fatty acids produced in the seed oil. Inaddition, the transformed seeds demonstrate altered polyunsaturatedfatty acid compositions as the result of the antisense KASI expression,such as seen in the decreases of linoleic (C18:2) and linolenic (C18:3)observed in the seed oil of plants containing elevated oleic acid.

Thus, using the methods of the invention, seeds are provided whichproduce an altered fatty acid composition and yield a vegetable oilwhich has increased oleic acid content and decreased linoleic andlinolenic acid content. Thus, the transformed seed can provide a sourceof modified seed oil.

The constructs used in the methods of the present invention may alsofind use in plant genetic engineering applications in conjunction withplants containing elevated levels of oleate (18:1) fatty acids tofurther increase oleic acid levels. Such plants may be obtained byexpression of stearoyl-ACP desaturase sequences, such as those sequencesdescribed by Knutzon et al. (Proc. Nat. Acad. Sci. (1992) 89:2624-2628).In addition, plants containing increased levels of oleic acid may beobtained by expressing nucleic acid sequences to suppress endogenous Δ12desaturases. Such sequences are known in the art and are described inPCT Publication WO 94/11516. Increases in oleic acid may also beobtained by suppression of Δ12-desaturases and Δ-15 desaturases, such asthe methods taught in U.S. Pat. No. 5,850,026. Plants producing elevatedoleic acid content may also be obtained by conventional mutation andplant breeding programs. Such methods for mutation are known in the artand are described, for example, in U.S. Pat. No. 5,625,130.

In addition, the constructs and methods for increasing oleic acid inseed oil may also find use in plant genetic engineering applications inconjunction with plants containing decreased levels of linoleate (C18:2)fatty acids and/or linolenate (18:3). Such plants with elevated levelsof stearate and/or with decreased levels of linoleate and/or linolenatemay be obtained through genetic engineering, or by conventional mutationand plant breeding programs. For example, methods for increasingstearate content of a seed oil are known in the art and are describedfor the use of a thioesterase from mangosteen (Garcinia mangostana),Garm FatA1 (Hawkins and Kridl (1998) Plant Journal 13(6):743-752; andPCT Patent Application WO 96/36719.

Furthermore, the constructs and methods for increasing oleic acid inseed oil may also find use in plant genetic engineering applications inconjunction with plants containing increased amounts of medium chainfatty acids to further increase the medium chain fatty acid content ofthe resulting plants. Such plants with elevated levels of medium chainfatty acids may be obtained through genetic engineering, or byconventional mutation and plant breeding programs. Methods forincreasing medium chain fatty acids by genetic engineering are known inthe art, and are described for example in U.S. Pat. Nos. 5,455,167, and5,512,482 and in PCT Publication WO 98/46776.

Thus, recombinant constructs designed having the KASI sequence in areverse orientation for expression of an anti-sense sequence or use ofco-suppression, also known as “transwitch”, constructs find use in themethods of the present invention. Antisense methods are well known inthe art, and are described, for example, by van der Krol, et al. (1988)Biotechniques 6:958-976; Sheehy, et al. (1988) Proc. Natl. Acad. Sci.USA 85:8805-8809; Cannon, et al. (1990) Plant Molec Biol. 15:39-47.Methods for sense suppression are also well known in the art, and aredescribed, for example, by Napoli et al. (1990) Plant Cell 2:279-289;van der Krol, et al. (1990) Plant Cell 2:291-299; and Smith, et al.(1990) Mol. Gen. Genetics 224:477-481.

Other methods for the suppression of native expression of targetsequences are also known in the art, and include, but are not limitedto, nucleic acid molecules with RNA cleaving activity, referred to asribozymes (described in PCT Publication WO 97/10328), as well ascombinations of antisense and sense suppression, such as that taught byWaterhouse, et al. (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964.Thus, by suppression of the endogenous fatty acid biosynthesis system,for example by the methods of the present invention, a reduction in theamounts of phosphtidyl choline (also referred to as PC) may be obtained.Such reductions in PC result in a lower substrate level for furtherdesaturases leading to increased amounts of mono-unsaturated fattyacids.

Sequences found in an anti-sense orientation may be found in cassetteswhich at least provide for transcription of the sequence encoding thesynthase. By anti-sense is meant a DNA sequence in the 5′ to 3′direction of transcription which encodes a sequence complementary to thesequence of interest. It is preferred that an “anti-sense synthase” becomplementary to a plant synthase gene indigenous to the plant host. Anypromoter capable of expression in a plant host which causes initiationof high levels of transcription in all storage tissues during seeddevelopment is sufficient. Seed specific promoters may be desired.

In preparing the expression constructs, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate in the proper reading frame. Towardsthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resection, ligation, or the like may beemployed, where insertions, deletions or substitutions, e.g. transitionsand transversions, may be involved.

For the most part, the constructs will involve regulatory regionsfunctional in plants which provide for modified production of plant KASI, and modification of the fatty acid composition. The open readingframe, coding for the plant KAS I or functional fragment thereof will bejoined at its 5′ end to a transcription initiation regulatory regionsuch as the wild-type sequence naturally found 5′ upstream to thethioesterase structural gene, or to a heterologous regulatory regionfrom a gene naturally expressed in plant tissues. Examples of usefulplant regulatory gene regions include those from T-DNA genes, such asnopaline or octopine synthase, plant virus genes, such as CaMV 35S, orfrom native plant genes.

For such applications when 5′ upstream non-coding regions are obtainedfrom other genes regulated during seed maturation, those preferentiallyexpressed in plant embryo tissue, such as ACP, napin and β-conglycinin7S subunit (Chen et al., (1986), Proc. Natl. Acad. Sci., 83:8560-8564)transcription initiation control regions, as well as the Lesquerellahydroxylase promoter (described in Broun, et al. (1998) Plant Journal13(2):201-210 and in U.S. patent application Ser. No. 08/898,038) andthe stearoyl-ACP desaturase promoter (Slocombe, et al. (1994) PlantPhysiol. 104:1167-1176), are desired. Such “seed-specific promoters” maybe obtained and used in accordance with the teachings of U.S. Pat. No.5,420,034 having a title “Seed-Specific Transcriptional Regulation” andin Chen et al., (1986), Proc. Natl. Acad. Sci., 83:8560-8564.Transcription initiation regions which are preferentially expressed inseed tissue, i.e., which are undetectable in other plant parts, areconsidered desirable for fatty acid modifications in order to minimizeany disruptive or adverse effects of the gene product.

Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant KAS I or a convenienttranscription termination region derived from a different gene source,for example, the transcript termination region which is naturallyassociated with the transcript initiation region. The skilled artisanwill recognize that any convenient transcript termination region whichis capable of terminating transcription in a plant cell may be employedin the constructs of the present invention. As described herein,transcription termination sequences derived from DNA sequencespreferentially expressed in plant seed cells are employed in theexpression constructs of the present invention.

The method of transformation is not critical to the instant invention;various methods of plant transformation are currently available. Asnewer methods are available to transform crops, they may be directlyapplied hereunder. For example, many plant species naturally susceptibleto Agrobacterium infection may be successfully transformed viatripartite or binary vector methods of Agrobacterium-mediatedtransformation. In addition, techniques of microinjection, DNA particlebombardment, and electroporation have been developed which allow for thetransformation of various monocot and dicot plant species.

In developing the DNA construct, the various components of the constructor fragments thereof will normally be inserted into a convenient cloningvector which is capable of replication in a bacterial host, e.g., E.coli. Numerous vectors exist that have been described in the literature.After each cloning, the plasmid may be isolated and subjected to furthermanipulation, such as restriction, insertion of new fragments, ligation,deletion, insertion, resection, etc., so as to tailor the components ofthe desired sequence. Once the construct has been completed, it may thenbe transferred to an appropriate vector for further manipulation inaccordance with the manner of transformation of the host cell.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin,etc., complementation providing prototrophy to an auxotrophic host,viral immunity or the like. Depending upon the number of different hostspecies in which the expression construct or components thereof areintroduced, one or more markers may be employed, where differentconditions for selection are used for the different hosts. A number ofmarkers have been developed for use for selection of transformed plantcells, such as those which provide resistance to various antibiotics,herbicides, or the like. The particular marker employed is not essentialto this invention, one or another marker being preferred depending onthe particular host and the manner of construction.

As mentioned above, the manner in which the DNA construct is introducedinto the plant host is not critical to this invention. Any method whichprovides for efficient transformation may be employed. Various methodsfor plant cell transformation include the use of Ti- or Ri-plasmids,microinjection, electroporation, DNA particle bombardment, liposomefusion, or the like. In many instances, it will be desirable to have theconstruct bordered on one or both sides by T-DNA, particularly havingthe left and right borders, more particularly the right border. This isparticularly useful when the construct uses A. tumefaciens or A.rhizogenes as a mode for transformation, although the T-DNA borders mayfind use with other modes of transformation.

Various methods of transforming cells of soybean have been previouslydescribed. Examples of soybean transformation methods have beendescribed, for example, by Christou et al. U.S. Pat. Nos. 5,015,580 andby Hinchee et al. U.S. Pat. No. 5,416,011, the entireties of which areincorporated herein by reference.

Once a transgenic plant is obtained which is capable of producing seedhaving a modified fatty acid composition, traditional plant breedingtechniques, including methods of mutagenesis, may be employed to furthermanipulate the fatty acid composition. Alternatively, additional foreignfatty acid modifying DNA sequence may be introduced via geneticengineering to further manipulate the fatty acid composition.

One may choose to provide for the transcription or transcription andtranslation of one or more other sequences of interest in concert withthe expression of a plant stearoyl-ACP thioesterase in a plant hostcell. In particular, the reduced expression of stearoyl-ACP desaturasein combination with expression of a plant stearoyl-ACP thioesterase maybe preferred in some applications.

When one wishes to provide a plant transformed for the combined effectof more than one nucleic acid sequence of interest, typically a separatenucleic acid expression constructs will be provided for each. Theconstructs, as described above contain transcriptional ortranscriptional and translational regulatory control regions. Theconstructs may be introduced into the host cells by the same ordifferent methods, including the introduction of such a trait by theinclusion of two transcription cassettes in a single transformationvector, the simultaneous transformation of two expression constructs,retransformation using plant tissue expressing one construct with anexpression construct for the second gene, or by crossing transgenicplants via traditional plant breeding methods, so long as the resultingproduct is a plant having both characteristics integrated into itsgenome.

By decreasing the amount of stearoyl-ACP desaturase, an increasedpercentage of saturated fatty acids is provided. Using anti-sense,transwitch, ribozyme or some other stearoyl-ACP desaturase reducingtechnology, a decrease in the amount of β-ketoacyl-ACP synthaseavailable to the plant cell is produced, resulting in a higherpercentage of oleate fatty acids.

Of special interest is the production of triglycerides having increasedlevels of oleic acid. In addition, the production of a variety of rangesof oleate is desired. Thus, plant cells having lower and higher levelsof oleate fatty acids produced by the methods described herein arecontemplated. For example, fatty acid compositions, including oils,having a 65% level of oleate as well as compositions designed to have upto an approximate 78% level of oleate or other such modified fattyacid(s) composition are contemplated.

The seeds of the invention which have been transformed with theconstructs providing antisense KASI expression provide a source fornovel oil compositions. The use of such constructs, for example, resultsin substantial increases in oleic acid content in seed oil. Bysubstantial increase is intended an increase of oleic acid to at leastabout 65% of the total fatty acid species. Thus, the seeds of theinvention which have been transformed with a antisense KASI expressionconstruct provide a source for modified oils having a high oleic acidcontent. The oleic acid content in any seed can be altered by thepresent methods, even those seeds having a naturally high oleic acidcontent. Alteration of seeds having naturally high oleic acid contentsby the present methods can result in total oleic acid contents of ashigh as about 78%.

Importantly, there is also a decrease in linoleic and linolenic acidcontent. By decrease in linoleic fatty acid content is intended adecrease of the linoleic fatty acid species to less than about 15 molpercent of the total fatty acid species. By decrease in linolenic fattyacid content is intended a decrease in linolenic acid to about less than7 mol percent of the total fatty acid content of the seed oil. Thus, themethods of the invention result in oils which are more oxidativelystable than the naturally occurring oils. The modified oils of theinvention are low-saturate, high oleic and low linolenic. Furthermore,the present invention provides oils high in monounsaturated fatty acidswhich are important as a dietary constituent.

Based on the methods disclosed herein, seed oil can be modified toengineer an oil with a high oleic acid content. High oleic acid oilswould have a longer shelf life as both the oleic acid content would lendto stability.

The methods of the present invention comprising the use of plantexpression or transcription constructs having a plant β-ketoacyl-ACPsynthase as the DNA sequence of interest may be employed with a widevariety of plant life, particularly, plant life involved in theproduction of vegetable oils for edible and industrial uses. Mostespecially preferred are temperate oilseed crops. Plants of interestinclude, but are not limited to, rapeseed (Canola and High Erucic Acidvarieties), sunflower, safflower, cotton, Cuphea, soybean, peanut, flax,coconut and oil palms, and corn. Depending on the method for introducingthe recombinant constructs into the host cell, other DNA sequences maybe required. Importantly, this invention is applicable to dicotyledonand monocotyledon species alike and will be readily applicable to newand/or improved transformation and regulation techniques.

The oil having increased oleic acid content may be processed usingmethods well known in the art. Furthermore, the processed oil havingincreased oleic acid produced by the methods of the present inventionfind use in a wide variety of end uses, such as edible as well asindustrial uses. Such applications include, for example, salad oils,frying oils, cooking oils, spraying oils, and viscous-food productapplications. The oil obtained according to the methods of the presentinvention, due to the increased monounsaturated fatty acid and thereduction in polyunsaturated fatty acid content, has a greater oxidativestability, thus reducing the need for chemical modifications, such ashydrogenation.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1 Expression Constructs

A construct containing the Brassica campestris synthase factor B (alsoreferred to as KAS I) cDNA sequence (SEQ ID NO:1), pCGN3248 (describedin U.S. Pat. No. 5,475,099, the entirety of which is incorporated hereinby reference), is mutagenized to insert SmaI, BglII and SalI restrictionsites approximately 200 bases 3′ of the translation stop signal,resulting in pCGN3255. pCGN3255 is digested at the factor B cDNAinternal SalI site located approximately 140 bases in from the 5′ end ofthe cDNA and at the 3′ BglII site inserted by mutagenesis. The resultingsynthase factor B cDNA fragment is ligated into BglII and SalI digestedpCGN3223, the above described napin expression cassette, resulting inantisense construct pCGN3257. Thus, transcription of the Brassicasynthase factor B sequence from the napin promoter will result inproduction of an mRNA strand that is complementary to that of theendogenous Brassica synthase factor B gene.

The fragment containing the synthase gene in the expression cassette, 5′sequences/synthase/3′ sequences, can be cloned into a binary vector suchas described by McBride and Summerfelt (Pl. Mol. Biol. (1990)14:269-276) for Agrobacterium transformation. Other binary vectors areknown in the art and may also be used for synthase cassettes.

For example, the antisense Brassica synthase factor B construct in anapin expression cassette, pCGN3257 is digested with Asp718 (samerecognition sequence as KpnI) and cloned into Asp718 digested pCGN1578(McBride and Summerfelt, supra) yielding binary construct pCGN3259.

Transformed Brassica napus plants containing the above describedconstructs are obtained as described in Radke et al. (Theor. Appl.Genet. (1988) 75:685-694 and Plant Cell Reports (1992) 11:499-505).

Example 2 Fatty Acid Analysis

The fatty acid composition is analyzed from about 50 individual seedsfrom each of two lines. The fatty acid compositions are shown in FIG. 1.

The results of the fatty acid compositional analysis demonstrates thatsignificant increases in oleic acid (18:1) are obtained in the oil ofBrassica seed containing antisense KAS I expression constructs. Oleicacid levels of as high as at least 78 mol percent are obtained usingsuch constructs, for example in lines 3259-D12 (#100). Smaller increasesare also obtained, for example several lines are obtained which containover 70 mol percent oleic acid. Furthermore, the majority of the linesobtained contain greater than about 65 mol percent oleic acid.

Furthermore, reductions in the amount of polyunsaturated fatty acids areobtained in the oil of seeds from Brassica plants containing antisenseKASI expression constructs. Amounts of linoleic acid is decreased tobelow 15% of the total fatty acid species, and as low as about 7.8 mol %in at least one seed of 3259-D-12. The linolenic acid content in theseeds of Brassica plants containing antisense KASI expression constructsis reduced to less than about 6 mol percent, and in some lines linolenicacid content is reduced to about 5.1 mol percent. Total polyunsaturatedfatty acid levels of less than about 13 mol percent may be obtainedusing such constructs.

Example 3 Identification of Soybean β-ketoacyl-ACP Synthase I Sequences

In order to produce soybean lines with increased oleic acid content,additional KASI DNA sequences from soybean EST libraries are identified.Four EST sequences from soybean are identified which are related to theBrassica KASI sequence (U.S. Pat. No. 5,475,099) (SEQ ID NOs:2-5). NineEST sequences are also identified in soybean EST libraries which arerelated to the Brassica KASII sequence (SEQ ID NO:6-14).

To obtain the entire coding region corresponding to the soybean KAS IEST sequences, synthetic oligo-nucleotide primers are designed toamplify the 5′ and 3′ ends of partial cDNA clones containingacyltransferase related sequences. Primers are designed according to therespective soybean KAS I EST sequence and used in Rapid Amplification ofcDNA Ends (RACE) reactions (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) using the Marathon cDNA amplification kit (ClontechLaboratories Inc, Palo Alto, Calif.).

Once a DNA sequence corresponding to the entire coding sequence, variousportions of the sequence, or the entire coding sequence may be used toconstruct antisense expression vectors for use in transforming soybeanplants using methods as provided for in the present invention.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

1. A modified Brassica seed oil comprising an increased oleic acid leveland a decreased polyunsaturated fatty acid level, wherein said increasedoleic acid level is greater than 65.8% and less than 67.39% of totalfatty acid content, and said decreased polyunsaturated fatty acid levelis greater than 20.87 mol percent and less than 26.09 mol percent oftotal fatty acid content.
 2. The modified Brassica seed oil according toclaim 1, further comprising a decreased linoleic acid level.
 3. Themodified Brassica seed oil according to claim 1, further comprising adecreased linolenic acid level.
 4. The modified Brassica seed oilaccording to claim 2, wherein said decreased linoleic acid level rangesfrom 11 mol percent to 18 mol percent of total fatty acid content. 5.The modified Brassica seed oil according to claim 3, wherein saiddecreased linolenic acid level ranges from 5 mol percent to 8 molpercent of total fatty acid content.
 6. The modified Brassica seed oilaccording to claim 4, further comprising a decreased linolenic acidlevel.
 7. The modified Brassica seed oil according to claim 6, whereinsaid decreased linolenic acid level ranges from 5 mol percent to 8 molpercent of total fatty acid content.
 8. The modified Brassica seed oilaccording to claim 7, further comprising a low-saturate fatty acidlevel.
 9. The modified Brassica seed oil according to claim 1, furthercomprising a low-saturate fatty acid level.
 10. A modified Brassica seedoil comprising an increased oleic acid level and a decreasedpolyunsaturated fatty acid level, wherein said increased oleic acidlevel is greater than 67.91% and less than 69.37% of total fatty acidcontent, and said decreased polyunsaturated fatty acid level is greaterthan 21.26 mol percent and less than 24.26 mol percent of total fattyacid content.
 11. A modified Brassica seed oil comprising an increasedoleic acid level and a decreased polyunsaturated fatty acid level,wherein said increased oleic acid level is greater than 69.57% and lessthan 70.93% of total fatty acid content, and said decreasedpolyunsaturated fatty acid level is greater than 19.12 mol percent andless than 21.47 mol percent of total fatty acid content.
 12. A modifiedBrassica seed oil comprising an increased oleic acid level and adecreased polyunsaturated fatty acid level, wherein said increased oleicacid level is greater than 71.23% and less than 73.99% of total fattyacid content, and said decreased polyunsaturated fatty acid level isgreater than 17.21 mol percent and less than 20.08 mol percent of totalfatty acid content.
 13. A modified Brassica seed oil comprising an oleicacid level and a polyunsaturated fatty acid level selected from thegroup consisting of: (a) an oleic acid level of 72.33 and apolyunsaturated fatty acid level of 17.21, (b) an oleic acid level of72.79 and a polyunsaturated fatty acid level of 17.25, (c) an oleic acidlevel of 73.08 and a polyunsaturated fatty acid level of 17.03, (d) anoleic acid level of 73.95 and a polyunsaturated fatty acid level of17.6, (e) an oleic acid level of 73.79 and a polyunsaturated fatty acidlevel of 16.38, and (f) an oleic acid level of 73.99 and apolyunsaturated fatty acid level of 17.84.